Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord

Gizelda T B Casella, Mary B Bunge, Patrick Wood

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Methods that facilitate the accurate counting of specific neural cell types would be of substantial value in evaluating the efficacy of treatments applied to spinal cord injury. This report describes reliable procedures for identification of neurons, oligodendrocytes, astrocytes, endothelial cells and inflammatory cells (neutrophils and activated macrophage/microglial cells) in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord. Antigen retrieval techniques (enzymatic and thermal) were used to improve antibody access to masked epitopes. To decrease background immunofluorescence and autofluorescence of hemoglobin, the tissue sections were pretreated with 0.1% sodium borohydride in PBS (30 min), followed by 1-5 min incubation in 0.5% Sudan black in 70% ethanol. Commercially available techniques to amplify the primary signal such as tyramide signal amplification (TSA) and avidin/biotin/peroxidase/ DAB/nickel/cobalt amplification (ABP/DABA) were also tested. Hoechst 33342 nuclear staining was used to indicate cell location, number, and integrity, thereby avoiding misidentification of cells. The best antibodies were: anti-NeuN antibody for neurons, anti-S100 for astrocytes, and anti-S100 and APC-7 antibodies in combination for oligodendrocytes, anti-laminin (LN) for endothelial cells, and ED1 antibody for activated macrophages and microglia. Amplification of the primary signal with TSA or ABP/DABA was also found to be beneficial.

Original languageEnglish
Pages (from-to)1-11
Number of pages11
JournalJournal of Neuroscience Methods
Volume139
Issue number1
DOIs
StatePublished - Oct 15 2004

Fingerprint

Paraffin
Spinal Cord
Endothelial Cells
Avidin
Antibodies
Oligodendroglia
Biotin
Cobalt
Nickel
Astrocytes
Peroxidase
Macrophages
Neurons
Sudan
Microglia
Laminin
Spinal Cord Injuries
Fluorescent Antibody Technique
Epitopes
Anti-Idiotypic Antibodies

Keywords

  • Antigen retrieval
  • Autofluorescence
  • Hoechst staining
  • Laminin
  • Signal amplification

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord. / Casella, Gizelda T B; Bunge, Mary B; Wood, Patrick.

In: Journal of Neuroscience Methods, Vol. 139, No. 1, 15.10.2004, p. 1-11.

Research output: Contribution to journalArticle

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